GSM6318832: ds20170407-334; Homo sapiens; RNA-Seq
Retinae were dissected from fetal eyes in ice-cold PBS, dissociated in 2 ml 10 u/ml papain at 37o C for 10 min, followed by gentle pipetting with a 1,000 ul tip to dissociate larger pieces every 5-10 min (at 37o C for an additional 20-30 min total) to obtain a mostly single-cell suspension. ~ 2 volumes of retina culture media [IMDM, 10% fetal bovine serum (FBS), 0.28 u/ml insulin, 55 uM β-mercaptoethanol, 1% by volume penicillin-streptomycin] was used to halt dissociation. Cells were centrifuged at 300 x g for 10 min, resuspended for counting, then recentrifuged and resuspended at 10,000 cell/ul in PBS with 5% FBS and CD133-PE, CD44-FITC, and CD49b FITC antibodies. After 1 h at room temperature, samples were diluted in PBS + 5% FBS, centrifuged, and resuspended in PBS with 1% FBS and 10 ug/ml DAPI. Forward and side scatter and CD133+/CD44,CD49b-low gating was used to enrich for cone photoreceptors. Single cells were either i) directly collected from FACS into 1.2 ul lysis buffer droplets on a parafilm-covered glass slide and transferred to low-adherence tubes for subsequent processing or ii) collected in bulk for isolation with the Fluidigm C1 microfluidics system. Full-length single cell cDNA transcripts were generated using SMARTer (Sequencing #1- 2) or SMART-SeqV4 chemistry (Sequencing #3 - 6) from Clontech (now Takara Bio) at ~ 0.1x scale using a Mantis liquid handler. Library preparation was performed using the Nextera XT DNA Library Preparation Kit at reduced volume using Mantis liquid handler and otherwise as per the manufacturer instructions.